1. Field of the Invention
This invention relates to chemiluminescent, enzymatically activatable, xanthan-esters and hydroxy acridan esters which are substrates for horseradish peroxidase. This invention further relates to the incorporation of these xanthan-esters and acridans in immunoassays, chemical assays and nucleic acid probe assays to permit an analyte--the chemical or biological substance whose presence, amount or structure is being determined--to be identified or quantified. Assay methods may be used to detect and quantitate various biological molecules including haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting respectively.
2. Description of Related Art
The detection and quantitation of biological molecules has been accomplished with several types of "labels" including radioisotopic, enzymatic, and phosphorescent/fluorescent. Typically, a detection method employs at least one analytical reagent that binds to a specific target macromolecular species or hapten and produces a detectable signal. These analytical reagents typically have two components: (1) a probe macromolecule, for example, an antibody or oligonucleotide, that can bind a target molecule with a high degree of specificity and affinity, and (2) a detectable label, such as a radioisotope or covalently linked fluorescent dye molecule. In general, the binding properties of the probe macromolecule define the specificity of the detection method, and the detectability of the associated label determines the sensitivity of the detection method. The sensitivity of detection is in turn related to both the type of label employed and the quality and type of equipment available to detect it.
Radioisotopic labels have several disadvantages, such as potential health hazards, difficulty in disposal, special licensing requirements and instability (radioactive decay and radiolysis).
Recently, numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Examples of such non-radioactive labels include: (1) enzymes that catalyze conversion of a chromogenic substrate to an insoluble, colored product (e.g. alkaline phosphatase, .beta.-galactosidase, horseradish peroxidase) or catalyze a reaction that yields a fluorescent or luminescent product, and (2) direct fluorescent labels (e.g. fluorescein, isothiocyanate, rhodamine, Cascade blue), which absorb electromagnetic energy in a particular absorption wavelength spectrum and subsequently emit visible light at one or more longer (i.e. less energetic) wavelengths.
Fluorescent labels do not offer the signal amplification advantage of enzyme labels, but they do possess significant advantages which have resulted in their widespread adoption in immunocytochemistry. Fluorescent labels typically are small organic dye molecules, such as fluorescein, Texas Red, or other rhodamines, which can readily be conjugated to probe molecules, such as immunoglobulins or Staph. aureus Protein A. The fluorescent molecules (fluorophores) can be detected by illumination with light of an appropriate excitation frequency and the resultant spectral emissions can be detected by electro-optical sensors or light microscopy.
Methods based on enzyme-linked analytes offer the best sensitivity since a single enzyme molecule typically has a persistent capacity to catalyze the transformation of a chromogenic substrate into detectable product. With appropriate conditions and incubation time, a single enzyme molecule can produce a large amount of product and hence yield considerable signal amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Chemiluminescent compounds which have been used in the prior art include aminophthalhydrazides, acridans, acridinium esters and dioxetanes. U.S. Pat. No. 5,593,845 discloses chemiluminescent N-alkylacridancarboxylate derivatives which allow the production of light from the acridan by reaction with a peroxide and a peroxidase. U.S. Pat. No. 5,686,258, discloses that the above mentioned reaction can be enhanced by the addition of a phenolic enhancer. U.S. Pat. No. 5,670,644, discloses improved acridan compounds which, upon reaction with a peroxidase enzyme and a peroxide compound, are converted into a more persistent, intermediate acridinium compound, wherein the center ring is aromatic, which subsequently undergoes a rapid chemiluminescent reaction when the pH is raised. U.S. Pat. No. 5,679,803 discloses chemiluminescent 1,2-dioxetanes which can be triggered to decompose and chemiluminesce by either enzymatic triggering agents or chemical triggering agents.
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. Amino-substituted cyclic phthalhydrazides such as luminol and isoluminol react with H.sub.2 O.sub.2 and a peroxidase enzyme catalyst (such as horseradish peroxidase) under basic conditions with emission of light. This reaction has been used as the basis for analytical methods for the detection of H.sub.2 O.sub.2 and for the peroxidase enzyme. Various enhancers have been employed in conjunction with the use of luminol to increase the intensity of light emitted. These include D-luciferin, p-iodophenol, p-phenylphenol and 2-hydroxy-9-fluorenone. A more complete list of peroxidase enhancers useful with compounds of this invention can be found in U.S. Pat. No. 5,206,149 (Oyama, et al.), which is incorporated by reference. Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are available.
Chemiluminescent substrates known in the art do not permit full advantage to be taken of the beneficial properties of horseradish peroxidase in analysis mainly due to sensitivity limitations. A substrate which permits the detection of lower amounts of enzyme is needed to enable the use of peroxidase conjugates in applications requiring ultrasensitive detection. Specifically, substrates are required which generate higher levels of chemiluminescence without an accompanying increase in the background or non-specific chemiluminescence. The increased chemiluminescence may be accomplished via either a higher maximum intensity or a longer duration than compounds known in the art.